The manifold biological effects of small peptides such as hormones and antibiotics warrant the study of the interaction of cells and peptides. Surprisingly little is known about the passage of peptides through membranes of eucaryotic cells, although the transport of oligopeptides into some bacteria has been examined extensively. We propose to study the structural specificities of peptide transport systems in procaryotic and eucaryotic microorganisms. Concurrently, the role of cellular peptidases in peptide metabolism will be evaluated. We aim to isolate macromolecular components of peptide transport systems from different cell types and to characterize them in detail. The eucaryotic microorganism selected for study is Saccharomyces cerevisiae. This yeast has an intracellular composition considerably more complex than that of bacteria. It can be used, therefore, to determine the role of subcellular organelles, such as vacuoles, in peptide transport in a eucaryote. Mutants of Salmonella typhimurium deficient in peptidases necessary for the hydrolysis of X-Pro bonds will be used as an example of a procaryote. Such peptidaseless mutants will accumulate certain dipeptides and oligopeptides intact and should be an optimum system for studying the kinetics and energetics of peptide uptake. Peptide transport will be followed primarily using direct assays with radioactive di- and oligopeptides. These substrates will be prepared by well-known procedures of peptide synthesis. Knowledge of the structural requirements of the peptide transport system(s) will be applied to the design of irreversible inhibitors of peptide uptake in the above microorganisms. Such affinity labels will be used to covalently insert radioactive or chromogenic markers into components of the peptide transport system and will thus aid in their isolation. In addition di-and oligopeptides will be bound to both insoluble and soluble macromolecules in attempts to isolate transport components using affinity chromatography techniques. After isolation, transport components will be characterized with biophysical methods. Information from this study should provide insights into the nature of the interaction of peptides with different cell membranes.